Bottom Line:
The AFP-Cre/LoxP-shRNA system target Atg5 gene could significantly sensitize MHCC97L/PLC cells to sorafenib-induced apoptosis in vitro and tumor growth suppression in vivo.An efficient HCC tissue-specific RNAi system (AFP-Cre/LoxP-shRNA) was successfully established.The system provides a usable tool for HCC-specific RNAi therapy, which may serve as a new treatment modality for HCC.

Background: RNA interference (RNAi) has recently emerged as a potential treatment modality for hepatocellular carcinoma (HCC) therapy, but the lack of cellular targets and sustained efficacy limits its application. The purpose of this study is to develop an HCC tissue-specific RNAi system and investigate its possibility for HCC treatment.

Methods: Two different HCC-specific RNAi systems in which therapeutic miRNA or shRNA against target gene (Beclin 1) was directly or indirectly driven by alpha-fetoprotein promoter (AFP-miRNA and AFP-Cre/LoxP-shRNA) were constructed. Human HCC cell lines (HepG2, Hep3B and HCCLM3) and non-HCC cell lines (L-02, Hela and SW1116) were infected with the systems. The effectiveness and tissue-specificity of the systems were examined by Q-PCR and western blot analysis. The efficacy of the systems was further tested in mouse model of HCC by intravenous or intratumoral administration. The feasibility of the system for HCC treatment was evaluated by applying the system as adjuvant therapy to enhance sorafenib treatment. An AFP-Cre/LoxP-shRNA system targeting Atg5 gene (AFP-Cre/LoxP-shRNA-Atg5) was constructed and its efficacy in sensitizing HCC cells (MHCC97L/PLC) to sorafenib treatment was examined by apoptosis assay in vitro and tumorigenesis assay in vivo.

Results: The AFP-miRNA system could silence target gene (Beclin 1) but required a high titer which was lethal to target cells. The AFP-Cre/LoxP-shRNA system could efficiently knockdown target gene while maintain high HCC specificity. Intratumoral injection of the AFP-Cre/LoxP-shRNA system could efficiently silence target gene (Beclin 1) in vivo while intravenous administration could not. The AFP-Cre/LoxP-shRNA system target Atg5 gene could significantly sensitize MHCC97L/PLC cells to sorafenib-induced apoptosis in vitro and tumor growth suppression in vivo.

Conclusions: An efficient HCC tissue-specific RNAi system (AFP-Cre/LoxP-shRNA) was successfully established. The system provides a usable tool for HCC-specific RNAi therapy, which may serve as a new treatment modality for HCC.

pone-0053072-g005: Application of AFP-Cre/LoxP-shRNA targeting Atg5 gene for enhancement of sorafenib treatment.(A) Construction of the AFP-Cre/LoxP-shRNA system targeting Atg5 gene (AFP-Cre/LoxP-shRNA-Atg5). Q-PCR and western blot analysis showed that it could efficiently knockdown Atg5 gene. (B) Combination of AFP-Cre/LoxP-shRNA-Atg5 infection and sorafenib treatment induced a significantly increase in apoptosis of HCC cells (MHCC97L and PLC) as compared to sorafenib treatment alone. Apoptosis was quantified by annexin V/PI FCM analysis. (C) Q-PCR and western blot analysis showed that intratumoral injection of the AFP-Cre/LoxP-shRNA-Atg5 efficiently silenced Atg5 gene in vivo. (D) The AFP-Cre/LoxP-shRNA-Atg5 significantly enhanced sorafenib-induced suppression of tumorigenicity of MHCC97L cells in vivo. The tumor weight of mice receiving intratumoral injection of the AFP-Cre/LoxP-shRNA-Atg5 combined with sorafenib was significantly lower than that of mice subjected to sorafenib alone. (Cre+LoxP: infection of AFP-Cre/LoxP-shRNA-Atg5; AFP-Cre and LoxP-shRNA: infection of AFP-Cre or LoxP-shRNA alone). (*P<0.05, **P<0.01).

Mentions:
Our previous study showed that silencing of Atg5 gene could enhance sorafenib-mediated lethality [18]. We next used the Atg5 as target gene to validate the effectiveness of the AFP-Cre/LoxP-shRNA system and test the value of the system for HCC treatment. The AFP-Cre/LoxP-shRNA system targeting Atg5 (AFP-Cre/LoxP-shRNA-Atg5) was successfully constructed. Q-PCR and western blot analysis showed that the AFP-Cre/LoxP-shRNA-Atg5 could efficiently downregulate Atg5 in AFP-producing cells (MHCC97L and PLC) (Fig. 5A). We then examined whether the AFP-Cre/LoxP-shRNA-Atg5 could sensitize HCC cells to sorafenib treatment. The efficacy of the AFP-Cre/LoxP-shRNA-Atg5 was firstly examined in vitro. Apoptosis assay showed that the apoptotic rate of cells (MHCC97L and PLC) treated with AFP-Cre/LoxP-shRNA-Atg5 plus sorafenibwas significantly higher than that of cells receiving sorefenib alone (all P<0.05) (Fig. 5B). To further evaluate whether the AFP-Cre/LoxP-shRNA system can be translated into a practical therapeutic modality, the AFP-Cre/LoxP-shRNA-Atg5 was next tested in vivo. A nude mouse model of HCC was established using MHCC97L cells and the AFP-Cre/LoxP-shRNA-Atg5 was intratumorally administrated. Q-PCR and western blot analysis showed that the AFP-Cre/LoxP-shRNA-Atg5 could efficiently downregulate Atg5 in vivo (Fig. 5C). The average tumor weight of mice receiving AFP-Cre/LoxP-shRNA-Atg5 plus sorafenib (1.04±0.33) was significantly lower than that of mice subjected to sorafenib alone (1.42±0.29) (P = 0.039) (Fig. 5D). These results indicated that the AFP-Cre/LoxP-shRNA-Atg5 could enhance sorafenib treatment.

pone-0053072-g005: Application of AFP-Cre/LoxP-shRNA targeting Atg5 gene for enhancement of sorafenib treatment.(A) Construction of the AFP-Cre/LoxP-shRNA system targeting Atg5 gene (AFP-Cre/LoxP-shRNA-Atg5). Q-PCR and western blot analysis showed that it could efficiently knockdown Atg5 gene. (B) Combination of AFP-Cre/LoxP-shRNA-Atg5 infection and sorafenib treatment induced a significantly increase in apoptosis of HCC cells (MHCC97L and PLC) as compared to sorafenib treatment alone. Apoptosis was quantified by annexin V/PI FCM analysis. (C) Q-PCR and western blot analysis showed that intratumoral injection of the AFP-Cre/LoxP-shRNA-Atg5 efficiently silenced Atg5 gene in vivo. (D) The AFP-Cre/LoxP-shRNA-Atg5 significantly enhanced sorafenib-induced suppression of tumorigenicity of MHCC97L cells in vivo. The tumor weight of mice receiving intratumoral injection of the AFP-Cre/LoxP-shRNA-Atg5 combined with sorafenib was significantly lower than that of mice subjected to sorafenib alone. (Cre+LoxP: infection of AFP-Cre/LoxP-shRNA-Atg5; AFP-Cre and LoxP-shRNA: infection of AFP-Cre or LoxP-shRNA alone). (*P<0.05, **P<0.01).

Mentions:
Our previous study showed that silencing of Atg5 gene could enhance sorafenib-mediated lethality [18]. We next used the Atg5 as target gene to validate the effectiveness of the AFP-Cre/LoxP-shRNA system and test the value of the system for HCC treatment. The AFP-Cre/LoxP-shRNA system targeting Atg5 (AFP-Cre/LoxP-shRNA-Atg5) was successfully constructed. Q-PCR and western blot analysis showed that the AFP-Cre/LoxP-shRNA-Atg5 could efficiently downregulate Atg5 in AFP-producing cells (MHCC97L and PLC) (Fig. 5A). We then examined whether the AFP-Cre/LoxP-shRNA-Atg5 could sensitize HCC cells to sorafenib treatment. The efficacy of the AFP-Cre/LoxP-shRNA-Atg5 was firstly examined in vitro. Apoptosis assay showed that the apoptotic rate of cells (MHCC97L and PLC) treated with AFP-Cre/LoxP-shRNA-Atg5 plus sorafenibwas significantly higher than that of cells receiving sorefenib alone (all P<0.05) (Fig. 5B). To further evaluate whether the AFP-Cre/LoxP-shRNA system can be translated into a practical therapeutic modality, the AFP-Cre/LoxP-shRNA-Atg5 was next tested in vivo. A nude mouse model of HCC was established using MHCC97L cells and the AFP-Cre/LoxP-shRNA-Atg5 was intratumorally administrated. Q-PCR and western blot analysis showed that the AFP-Cre/LoxP-shRNA-Atg5 could efficiently downregulate Atg5 in vivo (Fig. 5C). The average tumor weight of mice receiving AFP-Cre/LoxP-shRNA-Atg5 plus sorafenib (1.04±0.33) was significantly lower than that of mice subjected to sorafenib alone (1.42±0.29) (P = 0.039) (Fig. 5D). These results indicated that the AFP-Cre/LoxP-shRNA-Atg5 could enhance sorafenib treatment.

Bottom Line:
The AFP-Cre/LoxP-shRNA system target Atg5 gene could significantly sensitize MHCC97L/PLC cells to sorafenib-induced apoptosis in vitro and tumor growth suppression in vivo.An efficient HCC tissue-specific RNAi system (AFP-Cre/LoxP-shRNA) was successfully established.The system provides a usable tool for HCC-specific RNAi therapy, which may serve as a new treatment modality for HCC.

Background: RNA interference (RNAi) has recently emerged as a potential treatment modality for hepatocellular carcinoma (HCC) therapy, but the lack of cellular targets and sustained efficacy limits its application. The purpose of this study is to develop an HCC tissue-specific RNAi system and investigate its possibility for HCC treatment.

Methods: Two different HCC-specific RNAi systems in which therapeutic miRNA or shRNA against target gene (Beclin 1) was directly or indirectly driven by alpha-fetoprotein promoter (AFP-miRNA and AFP-Cre/LoxP-shRNA) were constructed. Human HCC cell lines (HepG2, Hep3B and HCCLM3) and non-HCC cell lines (L-02, Hela and SW1116) were infected with the systems. The effectiveness and tissue-specificity of the systems were examined by Q-PCR and western blot analysis. The efficacy of the systems was further tested in mouse model of HCC by intravenous or intratumoral administration. The feasibility of the system for HCC treatment was evaluated by applying the system as adjuvant therapy to enhance sorafenib treatment. An AFP-Cre/LoxP-shRNA system targeting Atg5 gene (AFP-Cre/LoxP-shRNA-Atg5) was constructed and its efficacy in sensitizing HCC cells (MHCC97L/PLC) to sorafenib treatment was examined by apoptosis assay in vitro and tumorigenesis assay in vivo.

Results: The AFP-miRNA system could silence target gene (Beclin 1) but required a high titer which was lethal to target cells. The AFP-Cre/LoxP-shRNA system could efficiently knockdown target gene while maintain high HCC specificity. Intratumoral injection of the AFP-Cre/LoxP-shRNA system could efficiently silence target gene (Beclin 1) in vivo while intravenous administration could not. The AFP-Cre/LoxP-shRNA system target Atg5 gene could significantly sensitize MHCC97L/PLC cells to sorafenib-induced apoptosis in vitro and tumor growth suppression in vivo.

Conclusions: An efficient HCC tissue-specific RNAi system (AFP-Cre/LoxP-shRNA) was successfully established. The system provides a usable tool for HCC-specific RNAi therapy, which may serve as a new treatment modality for HCC.